Speeding up temperature-responsive x reversed-phase comprehensive liquid chromatography through the combined exploitation of temperature and flow rate gradients

Comprehensive two-dimensional liquid chromatography (LC xLC) can provide enhanced resolving power and higher peak capacities for the separation of complex samples. The transfer of fractions of too high eluotropic strength from the first dimension, however, can lead to peak broadening. This process is related to the column dimensions, the flow rates, mobile phase compositions, and stationary phase compatibility. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica (poly(N-isopropylacrylamide), pNIPAAm), that exhibits a change in polarity upon modest variations in column temperature. Retention is thus modulated by temperature and not by organic solvents, allowing for the use of purely aqueous mo- bile phases. As these aqueous mobile phases depict a very low eluotropic strength on a reversed-phase column, it facilitates band refocusing at the second-dimension column head in TRLC xRPLC. One of the remaining obstacles of TRLC is the long analysis time. In this research, the potential of this column com- bination in terms of analyte refocusing will be exploited. First, it is shown that upwards flow gradients can be implemented in the first dimension of TRLC xRPLC. As the flow in the second dimension is main- tained at a constant level, a first-dimension flow gradient does not lead to impaired sensitivity and has no negative effects on the resulting peak size. Then, the novel combination of a downwards temperature gradient with an upwards flow gradient will be introduced to speed up the analyses further. Analysis time was, depending on the method used, reduced by 36-54%, as demonstrated by the analysis of mix- tures of food additives, phenolic compounds, and small molecule pharmaceuticals mimicking impurity analysis at a 0.05% level.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

Comprehensive two-dimensional liquid chromatography (LC xLC) is a method for separating complex samples that offers improved resolution and higher peak capacity. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica to modulate retention by temperature instead of organic solvents. This allows for the use of purely aqueous mobile phases and facilitates band refocusing. However, TRLC has the drawback of long analysis time.